MA1-980

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of c-Myc was performed by loading 40 µg of HeLa or NCCIT nuclear fractions (NF) onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% BSA/TBST for at least 1 hour. Membranes were probed with c-Myc Monoclonal Antibody, 9E10 (Product # MA1-980) at a dilution of 1:1000 overnight at 4°C on a rocking platform. Membranes were washed in TBS-0.1%Tween 20 and probed with a goat anti-mouse-IgG HRP secondary antibody at a dilution of 1:20,000 for at least one hour. Membranes were washed and chemiluminescent detection was performed using Super Signal West Dura (Product # 34075). Nuclear fractions were generated using the NE-PER kit (Product # 78833).

Supportive validation

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Experimental details: Western blot analysis of c-Myc was performed by loading 20 µg of 293T or 293T-c-myc whole cell lysate onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% BSA/TBST for at least 1 hour. Membranes were probed with c-Myc Monoclonal Antibody, 9E10 (Product # MA1-980) at a dilution of 1:1000 overnight at 4°C on a rocking platform. Membranes were washed in TBS-0.1%Tween 20 and probed with a goat anti-mouse-IgG HRP secondary antibody at a dilution of 1:20,000 for at least one hour. Membranes were washed and chemiluminescent detection performed using Super Signal West Dura (Product # 34075).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of c-Myc was performed by loading 25-100 ng recombinant Myc-tagged protein onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% BSA/TBST for at least 1 hour. Membranes were probed with c-Myc Monoclonal Antibody, 9E10 (Product # MA1-980) at a dilution of 1:1000 overnight at 4°C on a rocking platform. Membranes were washed in TBS-0.1%Tween 20 and probed with a goat anti-mouse-IgG HRP secondary antibody at a dilution of 1:20,000 for at least one hour. Membranes were washed and chemiluminescent detection was performed using Super Signal West Dura (Product # 34075).

Supportive validation

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Experimental details: Western blot was performed using Anti-Myc Tag Polyclonal Antibody (Product # MA1-980-1MG) by loading whole cell extracts of untransfected and transiently transfected HEK-293E lysates: untransfected, 40 µg (Lane 1), empty vector control, 40 µg (Lane 2), Myc-p65-V5, 40 µg (Lane 3), Myc-p65-V5, 20 µg (Lane 4), Myc-p65-V5, 10 µg (Lane 5), Myc-p65-V5, 5 µg (Lane 6), p65-HA, 60 µg (Lane 7) and 25 ng of Positope (Lane 8) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). A ~65 kDa band corresponding to Myc-p65-V5 was observed in HEK293E transfected lysates on probing with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP (Product # A28177, 1:4000 dilution). Positope (Product # R90050) is a 53 kDa recombinant protein consisting of multiple epitope tags but lacks HA tag, which has been used as a positive control for c-Myc tag detection. No cross-reactivity was seen with HA-tagged p65 expressing lysate.

Supportive validation

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Experimental details: Immunofluorescent analysis of c-Myc (green) in HEL 11.4 induced IPS cells grown for a few days on Matrigel-coated chamber slides. Cells fixed in 4% paraformaldehyde were permeabilized with 0.1% Triton X-100 for 15 minutes at room temperature. Cells were probed with a c-Myc monoclonal antibody (Product # MA1-980) at a dilution of 1:200 overnight at 4°C, washed with PBST, and incubated with a FITC-conjugated secondary antibody at a dilution of 1:100 for 1 hour at room temperature. Nuclei (blue) were stained with DAPI and cells were analyzed by fluorescence microscopy at 20X magnification.

Supportive validation

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Experimental details: Immunofluorescent analysis of c-Myc (green) in H9 embryonic stem cells grown for a few days on Matrigel-coated chamber slides. Cells fixed in 4% paraformaldehyde were permeabilized with 0.1% Triton X-100 for 15 minutes at room temperature. Cells were probed with a c-Myc monoclonal antibody (Product # MA1-980) at a dilution of 1:200 overnight at 4°C, washed with PBST, and incubated with a FITC-conjugated secondary antibody at a dilution of 1:100 for 1 hour at room temperature. Nuclei (blue) were stained with DAPI and cells were analyzed by fluorescence microscopy at 20X magnification.

Supportive validation

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Experimental details: Immunofluorescent analysis of Myc tag was performed using 70% confluent His-H3-Myc transfected HEK-293 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with Myc Tag Monoclonal Antibody (Product # MA1-980-1MG) at 1:100 dilution and Histone H3 antibody (Product # 711055) at 1:200 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight, and then labeled with Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647 (Product # A32728) and Goat anti-Rabbit IgG Alexa Fluor Plus 488 (Product # A32731) respectively at a dilution of 1:2000 for 45 minutes at room temperature. Panel a (Nuclei: Red) represents the Myc tag. Panel b (Nuclei: Green) represents Histone H3. Panel c (Nuclei: Blue) represents ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). Panel d represents the merged image showing the co-localization of nuclear signals in transfected cells. Panel e represents un-transfected HEK-293 cells. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

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Experimental details: Immunohistochemistry was performed on cancer biopsies of deparaffinized Human breast carcinoma tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:100 with a Mouse Monoclonal Antibody recognizing c-myc (Product # MA1-980) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry was performed on cancer biopsies of deparaffinized Human lung squamous carcinoma tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:100 with a Mouse Monoclonal Antibody recognizing c-myc (Product # MA1-980) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry was performed on normal biopsies of deparaffinized Human tonsil tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:50 with a Mouse Monoclonal Antibody recognizing c-myc (Product # MA1-980) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Supportive validation

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Supportive validation

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Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

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Experimental details: Fig 2 AtMORC4 and AtMORC7 form homomeric complexes in vivo . (A) Co-immunoprecipitation of AtMORC4-MYC with AtMORC4-FLAG in F1 plants (B) Co-immunoprecipitation of AtMORC7-MYC with AtMORC7-FLAG in F1 plants. (C) No interaction by co-immunoprecipitation between AtMORC7-MYC and AtMORC4-FLAG in F1 plants. (D) Table from immunoprecipitation followed by mass spectrometry (IP-MS) of FLAG tagged AtMORC4 and AtMORC7 plants showing peptides from themselves but not each other. NSAF = normalized spectral abundance factor.

Supportive validation

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Experimental details: Figure 3 Correlation Rtc4 and Gis2 protein expression in response to zinc status. Wild-type (BY4741) cells transformed with plasmid pRHGM were grown in LZM with the indicated zinc level for 16 h prior to immunoblot analysis. pRHGM expresses HA-tagged Rtc4 and myc-tagged Gis2. Pgk1 was used as a loading control and the asterisk marks a non-specific band. The location of size markers is indicated on the left side of the panel in kDa. The histograms show quantified results from three replicates compared to 100 uM zinc cultures as the replete (ZnR) condition. Error bars are +- 1 SD.

Supportive validation

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Experimental details: Figure 5 Mutagenesis analysis of RTC4 and GIS2 co-regulation. Wild-type (BY4741) cells were transformed with plasmids pRHGM, pRHGM mZRE , pRHGM Ter , or pGM were grown in grown in zinc-replete (R, LZM + 100 uM ZnCl 2 ) or deficient (D, LZM + 1 uM ZnCl 2 ) media prior to analysis. ( A ) Diagram of the plasmids used in this experiment. ( B ) Immunoblot analysis of Rtc4-HA, Gis2-myc, and Pgk1. ( C ) Northern blot analysis of RNA expression using HA tag and myc tag-specific probes. TAF10 was used as a loading control. The locations of the GIS2 and RTC4 SLT2 mRNAs are marked as well as the LLT RNA and truncated LLT RNA produced in the pRHGM Ter transformant. RTC4 SLT1 was not detectable in this experiment because of its low abundance. The asterisks mark two transcripts that arise from transcription initiation in the plasmid vector for pGM. The location of size markers is indicated on the left side of each panel in kDa (panel B) or kb (panel C).

Supportive validation

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Experimental details: Figure 7. PHF8 is upregulated in breast cancers. ( A ) IHC analysis of PHF8 expression in human breast cancer samples. Status of markers associated with locations and stages are listed for each tumor. Scale bar: 10 mum. ( B ) Summary of PHF8 immunohistochemical staining of 190 breast cancer, metastatic lymph nodes and adjacent normal tissues. ( C ) IHC analysis of the expression levels of PHF8 and MYC in 46 breast cancer samples. Representative images (original magnification, x400) from breast carcinoma samples at different stages are shown. The immunoreactive score (IRS) was determined by staining intensity and distribution. The correlation coefficient ( r ) and P values were analyzed as indicated (right panel).

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Experimental details: Fig. 9 Notch1 intracellular domain (NICD) generation is not affected in Syt1 KD PC12 cells. Parental and Syt1 KD PC12 cells were transfected with Myc-tagged, constitutively active Notch1 with deleted extracellular domain (NDeltaECD), which is an immediate substrate of the gamma-secretase. The cells were treated with DMSO or gamma-secretase inhibitor -- DAPT. Transfection with a plasmid encoding Notch1 intracellular domain (NICD) was used as a control. Western blot analysis shows no difference in the production of the Notch1 intracellular domain, as detected by anti-myc antibody, between parental and Syt1 KD PC12 cells. Data are presented as the NICD/NDeltaECD ratio; mean +- SEM, n = 4. Statistical significance was determined using unpaired student t -test. Syt1 synaptotagmin 1

Supportive validation

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Experimental details: FIGURE 2: beta-CTT promotes normal cell cycle progression. (A) Normalized doubling times for WT and CTT mutant cells. Values are the mean from at least four separate experiments. Error bars are SEM. ** p < 0.0001 determined by t test. (B) Duration of S/G2/M determined by measuring the time from bud emergence to separation in cells released from START. Dashed lines are the medians. WT, n = 617; tub2-430Delta, n = 561. (C) Time course of Pds1/securin levels in synchronized cells released from START. Cells expressing Pds1-13myc were collected at 15- min intervals, prepared for Western blots, and probed with myc antibodies. (D) Pds1-13myc signal at each time point normalized to t = 0. Values are averages from three experiments. Error bars are SEM.

Supportive validation

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Experimental details: Immunoprecipitation (IP) studies of Myc tag were performed with c-Myc Monoclonal Antibody (9E10) (Product # MA1-980) (Panel I, II) or NFkB p65 Monoclonal Antibody (Product # 33-9900) (Panel III, IV) using the Dynabeads® Protein G Immunoprecipitation Kit (Product # 10007D). Lane 1,2: Total cell extract of HEK-293E untransfected (UT) and transfected (T) with Myc-tagged NFkB p65 construct (7% of input) Lane 3,4: IP of UT and T HEK-293E lysate using Isotype-matched Mouse IgG (Product # 31903) Lane 5,6: IP of UT and T HEK-293E lysate with 5 µg of c-Myc Monoclonal Antibody (9E10) (Product # MA1-980) (Panel I, II) (Panel I, II) or NFkB p65 Monoclonal Antibody (Product # 33-9900) (Panel III, IV) IP was analyzed using western blot. Myc-tagged NFkB p65 was detected at ~65 kDa by probing the blot with NFkB p65 Monoclonal Antibody (Product # 33-9900) (0.5 µg/mL dilution) (Panel I, IV) or c-Myc Monoclonal Antibody (9E10) (Product # MA1-980) (1:1,000 dilution) (Panel II, III) and detected by chemiluminescence with Recombinant Protein G-HRP (Product # 101223) (1:500 dilution) using the iBright FL 1500 (Product # A44241). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

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Experimental details: Fig 5 Interdependency of SHREC components to associate with heterochromatin. (A) Schematic diagram of the left half of centromere 1. Position of the PCR product used to detect Clr3-myc in (B) and F-Chp2 in (C) is indicated by a thick bar. (B,C) ChIP assay showing Clr3-myc (B) or F-Chp2 (C) levels at centromeric dg . The values were normalized to input. Error bars indicate standard errors from three biological replicates. The strains used were PJ1323 (wt), PJ 1600 ( clr3-myc ), EO1001 ( clr3-myc , chp2 Delta), EO1002 ( clr3-myc , F-chp2-W199 ), EO1003 ( clr3-myc , clr1 Delta), EO1004 ( clr3-myc , clr2 Delta), PJ1566 ( F-chp2 ), EO1005 ( F-chp2-W199A ), EO1006 ( F-chp2 , clr1 Delta), EO1007 ( F-chp2 , clr1 Delta), and EO1006 ( F-chp2 , mit1 Delta).

Supportive validation

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Experimental details: Figure 6 ADSL overexpression induces succination and mTOR activation and increases c-MYC protein expression. ( A ) Proliferation capacity of DLD-1 control and ADSL -silenced cells treated with DMSO or fumarate (50 uM). ( B ) Immunoblot showing ADSL and c-MYC expression in the Caco-2 cells at 48 and 72 h post transfection. ( C ) Representative pictures of control and ADSL -overexpressing Caco-2 cells immunostained with c-MYC. Scale bars 20-50 um. ( D ) Immunoblot showing ADSL and c-MYC expression in CRC cell lines (left). Correlation (linear regression) between c-MYC (Y-axis) and ADSL (X-axis) levels of expression (relative to actin). ( E ) Immunoblot showing ADSL, c-MYC and phospho and total S6 expression in Caco-2 cells 48 h post-transfection. ( F ) Representative pictures of control and ADSL -overexpressed Caco-2 cells immunostained with phospho-S6 (p-S6). Scale bars 20-50 um. ( G ) Immunoblot showing ADSL and succination (2-SC) levels in Caco-2 cells 48 h post-transfection. ( H ) Immunoblot showing ADSL and succination (2-SC) levels (left) and KEAP1, NRF2, TOM20, total and phospho-S6 in control and ADSL -overexpressing Caco-2 cells treated with DMSO or fumarate (50 uM). ( I ) Schematic representation of ADSL-driven pro-oncogenic effects in CRC cells.

Supportive validation

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Experimental details: Fig. 3. IF-combined smRNA FISH analysis of MCPIP1 protein interaction with IER3 mRNA. (A) Representative fluorescence images of HepG2 cells transfected with empty vector (mock, upper panel) or with expression vectors coding for MCPIP1-MycHis (middle panel) or EGFP-MycHis (lower panel) and subsequently subjected to IF-combined smRNA FISH procedure (DAPI, nuclei; Myc, primary antibody: mouse anti-c-myc, secondary antibody: Alexa Fluor 488-conjugated donkey anti-mouse; EGFP, native fluorescence of EGFP-MycHis; IER3 , probe blend labeled with fluorescent dye Quasar 570). (B) Quantitative analysis of IF-combined smRNA FISH images of HepG2 cells transfected with empty vector (mock) or with expression vectors coding for MCPIP1-MycHis, MCPIP1(D141A)-MycHis or EGFP-MycHis. Column scatter graph presents all analyzed cells with mean number of IER3 mRNA particle count for each experimental variant. Error bars=s.e.m. Applied statistical method: one-way ANOVA with Bonferroni post hoc test. Numbers of analyzed cells: mock, n= 21, MCPIP1-MycHis, n= 17; MCPIP1(D141A)-MycHis, n= 12 and EGFP-MycHis, n= 7. ns, not significant; **** P

Supportive validation

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Experimental details: Figure 3 FLYWCH1 regulates certain beta-catenin/TCF4 target genes involved in the AML. ( A ) Western blot analysis of beta-catenin target gene expression in AML cell lines using antibodies against cyclin D1, c-Myc, c-Jun, and beta-actin loading control. Asterisks (*) indicate unspecific bands. ( B - F ) Effect of ( B ) FLYWCH1 gene expression modulation on: ( C ) c-JUN , ( D ) c-MYC , ( E ) LGR5 , and ( F ) FGF4 transcription activity. Data are mean +- SD ( n = 6; * p < 0.05; ** p < 0.01; *** p < 0.001).

Supportive validation

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Experimental details: Fig. 4 Dimerisation between HSI2 and HSL1 is dependent on PHD-like domain. Bimolecular fluorescence complementation (BiFC) analysis of protein-protein interactions between (a) HSI2mB3:HSI2mB3, (b) HSI2mB3:HSL1, (c) HSI2mPHD:HSI2mPHD and (d) HSI2mPHD:HSL1 using Nicotiana benthamiana leaves co-infiltrated with combinations of corresponding constructs. (e) Co-immunoprecipitation (Co-IP) showing interaction between HSI2mB3 and HSI2mB3 and (f) HSI2mB3 and HSL1. Total protein was extracted from agroinfiltrated tobacco leaves co-expressing 35S:HSI2mB3-GFP and 35S:HSI2mB3-myc , 35S:HSL1-GFP and 35S: HSI2mB3-myc , only 35S:HSI2mB3-GFP or only 35S:HSL1-GFP . HSI2mB3-GFP and HSL1-GFP was immunoprecipitated with anti-GFP antibody and immunoblots were probed with anti-c-myc antibody. (g) Yeast-two-hybrid interaction assays. Yeast cells transformed with indicated genes were selected on Double Dropout medium (DDO, lacking Leu and Trp) and quadruple dropout medium (QDO, lacking adenine, His, Leu and Trp) media.

Supportive validation

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Experimental details: Figure 5 IC 50 of alpha-naphthoflavone (ANF) for the inhibition of CYP1A1 enzyme expressed within yRD + and yRD - :: Bax cells. ANF's IC 50 for inhibition of CYP1A1 enzyme that was expressed in live cells. The cells that were used were from strains ( A ) yRD + (+CPR) and ( B ) yRD - :: Bax (-CPR). CYP1A1 enzyme expression was driven by the ethanol-inducible ADH2 promoter (ADH2p). In yRD - cells, Bax expression was driven by the GAL1 promoter (GAL1p), which was de-repressed in ethanol. Recombinant cells were grown for 18 h in YPD liquid medium containing glucose, allowing for around 6 h of induction and de-repression of ADH2p and GAL1p in ethanol (it takes ~12 h for glucose to be utilized and entirely converted to ethanol). Cells were harvested, washed in PBS-saline. The assay included 1 x 10 7 washed cells and 7-ethoxyresorufin (7-ER) as substrates. The x -axis represents the values of the concentration of ANF in uM (ranges from 0.00001 to 3 uM) that were incubated with cells for 10 min, the y -axis relative fluorescence units of the fluorescent product formed. The IC 50 values were calculated using the GraphPad Prism software. T. ANF's IC 50 for inhibition of CYP1A1 Supersomes (insect cell-derived CYP1A1-bearing microsomes; Corning-Gentest, Corning, NY, USA) is 18 nM []. ( C ) Western Blotting of lysates from yRD + , yRD - , and yRD-:: Bax cells expressing CYP1A1. Yeast cell lysates (5 ug of total cellular protein for monitoring CYP1A1 and actin; 50 ug for Bax) fractionated

Supportive validation

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Experimental details: Figure 1 Oncolytic Vaccinia virus (VV) and Semliki Forest virus (SFV) encoding the GD2 mimotope (GD2m) (A and B) Schematic illustration of VV Western Reserve (VVWR) and SFV constructs used in the study. (C) GD2m expression in NXS2 tumors injected with VV-dTK or VV-GD2m (i.t. ), visualized by immunohistochemical staining. Tumors were collected 2 days after virus injection, and the tumor tissue section was stained with anti-c-Myc antibody and DAPI. Scale bars, 50 mum. (D and E) GD2m expression in NXS2 tumors injected with SFV or SFV-GD2m (i.t.). Tumors were harvested 2 days after virus injection and single-cell suspension was prepared and stained with anti-c-Myc antibody. Representative histogram figures were shown. (F) Representative histogram showing binding of the anti-GD2 antibody to cells infected with VV-GD2m. B16-F10 cells were infected with VV-GD2m (MOI 0.1) and were collected after 48 h. Cells were stained with anti-GD2 antibody (clone 14G2a) and secondary goat anti-rabbit-AF647 antibody and analyzed by flow cytometry. NXS2 cells (naturally expressing GD2) were used as a positive control. VV-GD2m-infected B16-F10 cells stained with only secondary goat anti-rabbit-AF647 antibody were used as a negative control.

Supportive validation

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Experimental details: Fig. 3 a , d Measurement of ROS produced in the yeast strains Bax(LEU2):: eEF 1A1 ( a ), Bax(LEU2)::Bcl 2A1 ( d ) and Bax(LEU2):: -, a two-tailed paired sample t test show that there was significant difference (p < 0.05). b , e TUNEL assay comparing the nuclear DNA fragmentations in yeast strains Bax(LEU2):: eEF 1A1 ( b ), Bax(LEU2)::Bcl 2A1 ( e ) and Bax(LEU2):: -. The cells Bax(LEU2):: eEF 1A1 and Bax(LEU2)::Bcl 2A1 have lower nuclear DNA fragmentation compared to Bax(LEU2):: - cells. A two tailed paired sample t test show that there was significant difference (p < 0.05). c , f represent microscopic image of cells after tunnel assay, Bax(LEU2):: eEF 1A1 ( c ) and Bax(LEU2)::Bcl 2A1 ( f ). g Western blot to monitor the presence of Bax in yeast cells that express only Bax and in cells that co-express Bax together with eEF-1A1 and, Bcl-2A1. 10 ug of total cellular proteins, in cell lysates, were loaded on to each lane and the blot was probed with an antibody that recognizes the c-myc-tag, Actin, & HA-tag. The plasmid pRS305/GAL1p-h_Bax-MS has restriction sites that cut the plasmid only once (Supporting information Fig. 1). The human Bax gene contains, at the 3'-end, a sequence that codes for the c-myc tag so that expressed protein can be monitored with an antibody that recognizes the c-myc epitope at the C-terminus of Bax protein. Plasmid pSYE239/eEF1A1-HA (Supporting information Fig. 2) is an episomal 2u-plasmid encoding HA-tagged eEF1A1 gene. The plasmid pSYE239/Bcl-2A1-HA i

Supportive validation

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Experimental details: Figure 3 Reactive oxygen species (ROS) measurement, terminal deoxynucleotidyl transferase (TdT)-facilitated dUTP nick end labelling (TUNEL) assay, and Western blotting. ( A ) Measurement of ROS produced in the yeast strains Bax(LEU2) + VAMP3, Bax(LEU2) + Bcl xL, and Bax(LEU2) -. A two way ANOVA test shows that there was a significant difference ( p < 0.05) between the control strain and each of Bax + VAMP3 and Bax + Bcl-xL, but there was no significant difference ( p > 0.05) between Bax + VAMP3 and Bax + Bcl-xL. The data represent the mean +- S.D. of three independent experiments. ( B ) TUNEL assay comparing the nuclear DNA fragmentations in yeast strains Bax(LEU2)VAMP3, Bax(LEU2)Bcl xL, and Bax(LEU2). The cells Bax(LEU2)VAMP3 and Bax(LEU2)Bcl xL have lower nuclear DNA fragmentation compared to Bax(LEU2)-cells. A two way ANOVA test shows that there was a significant difference ( p < 0.05) between the control strain and each of Bax + VAMP3 and Bax + Bcl-xL, but there was no significant difference ( p > 0.05) between Bax + VAMP3 and Bax + Bcl-xL. The data represent the mean +- S.D. of three independent experiments. ( C ) Represent the microscopic image of cells after tunnel assay, Bax(LEU2)VAMP3 and Bax(LEU2)Bcl xL. The left images are representative of cells under fluorescent light, showing dead cells (red) (high nuclear fragmentation) . ( D ) Western blot to monitor the presence of Bax in yeast cells that express only Bax and in cells that coexpress Bax together with VAMP3 an

Supportive validation

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Experimental details: Figure 10 Rescue of Bax induced block of cell growth by untagged alpha-synuclein (1-3 copies) on GAL1p and alpha-synuclein-HA (1-3 copies) on MET25p ( A ) Growth on solid agar minimal medium plates of yeast transformants bearing one (1), two (2) or three (3) copies of the alpha-synuclein gene under the control of MET25 promoter (MET-alpha-syn-HA) and Bax gene under the control of GAL1 promoter (Bax). ( B ) Growth of yeast strains containing 1, 2 and 3 copies of alpha-synuclein-HA (MET25p) with a copy of Bax in a minimal liquid medium (SG + 0.1% glucose) along with controls. There was a significant difference among the three yeast strains with their respective controls ( P 0.05), ANOVA test also revealed that there was no significant difference between one and two copies ( P >0.05), but there was a significant difference between two and three copies ( P

Supportive validation

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Experimental details: Figure 2 Effect of AR singling on E2F1 , MYC , and TERT expression. (A , D) MYC was upregulated in the group with high AR signaling signature score ( SOX9 , RAN , TNK2 , EP300 , PXN , NCOA2 , AR , NRIP1 , NCOR1 , and NCOR2 ). MYC was significantly positively correlated with AR signaling score. (B , E) E2F1 was downregulated in AR signaling high group compared to AR signaling low group. E2F1 was negatively correlated with AR signaling score. (C, F) No significant correlation was observed for TERT upon the AR signaling signature in the TCGA cohort. The Z score sum of the ten-gene AR signaling signature was stratified based on the median score. p- values were calculated using Mann-Whitney U test. (G-I) mRNA levels of E2F1 (G) , TERT (H) , and MYC (I) were elevated in human benign, primary, and metastasis PCa in the Grasso cohort. p- values were calculated using Kruskal-Wallis test. (J) Western blot showing protein levels of TERT, E2F1, and MYC in LNCaP cells with hormone-deprived medium for the indicated days. GAPDH was used as a loading control. (K-O) Expression correlation of MYC with E2F1 in human prostate tissues. Scatter plot showing the direct correlation between MYC with E2F1 expression in the CPGEA cohort ( n = 272) (K) , Barwick cohort ( n = 139) (L) , Yu cohort ( n = 112) (M) , Grasso cohort ( n = 122) (N) Singh cohort ( n = 102) (O) , and Tomlins ( n = 101) (P) . (Q, R) Western blot showing the protein level of MYC in LNCaP cells overexpressing V5 tagged E2F1 (P) and

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Experimental details: FIGURE 1 HDAC6 co-localizes with DPR pathology in c9FTD/ALS. (A) Representative images of immunohistochemical analysis of HDAC6 in frontal cortex (top panels) and hippocampus (bottom panels) from control, sporadic FTD/ALS, or c9FTD/ALS patients ( n = 5 per group). Arrowheads indicate HDAC6 inclusions. Scale bars, 20 mum. (B) Triple-immunofluorescence staining for HDAC6, poly (GR), and either poly (GA) (top panel) or poly (GP) (bottom panel) in the hippocampus of c9FTD/ALS patients. Scale bars, 10 mum. (C) Quantitative analysis of the percentage of HDAC6-positive inclusions that co-localize with poly (GA), poly (GP) and poly (GR) in the hippocampus of c9FTD/ALS patients ( n = 5). Data are presented as mean +- SEM, **** p < 0.0001, one-way ANOVA, Tukey's multiple-comparison test. (D) HEK293T cells were transfected with myc-HDAC6 and GFP, GFP-(GA) 50 , or GFP-(GP) 47 . GFP and myc-HDAC6 levels were evaluated in cell lysates by immunoblotting (left panel), with GAPDH used to control for protein loading. Myc-HDAC6 or GFP were immunoprecipitated from cell lysates, followed by immunoblotting for myc or GFP (right panel).

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Experimental details: Fig. 6 CARP3 is required for acid sensing in vitro and for establishment of infection in flies. a Merged EP and GPEET signals of Lister 427 wt and PDEB1 knockout (KO). b Merged EP and GPEET signals of 427 Cas9 parental line, CARP3 knockout and CARP3-HA addback. Upper panel: untreated. Lower panel: exposed to acid and alkali. c Teneral flies were infected with wild-type Lister 427 procyclic forms (WT), the CARP3 double knockout (CARP3-KO) and the addback (AB). Dissections were performed 13-14 days post infection. Total numbers are derived from two replicates per cell line (Supplementary Table 2 ). The intensity of midgut infections was scored as heavy, intermediate or weak as described . The p-value is shown for Fisher's exact test, two-sided ( p -value = 2.0117 -10 ). d Western blot analysis of immunoprecipitations with anti-HA and anti-myc antibodies (representative blot from three independent experiments). Myc-tagged ACP3 and ACP5 were expressed independently in the CARP3-HA background. The parental line 427 Cas9 was used as a control. Input samples have 40 times fewer cell equivalents than the immunoprecipitated samples. IP: immunoprecipitation with anti-HA or anti-myc antibodies. e Merged EP and GPEET signals of the 427 Cas9 parental line and 3 independent ACP5 single allele knockouts; panels as in ( b ). f Myc-tagged ACP5 is localised to the flagellar tip in a cell line co-expressing CARP3-HA. Immunofluorescence was performed with an anti-myc antibody (red). DNA was stai

Supportive validation

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Experimental details: Figure 4. The rescue of mesodermal C&E by endodermal expression of GFP-Gpc4 is mediated in part by Wnt5b and Wnt11f2. (A and B) Coimmunoprecipitation experiments were performed using lysates from HEK cells transfected with Myc or Myc-Mmp14b (negative controls), or Wnt5b-Myc or Wnt11f2-Myc and Flag-Gpc4 or Flag-JNK (negative controls) using anti-C-MYC antibody coupled to protein G magnetic beads. Western blotting of in both pellets (IP) and cell lysates (Lysate) was performed using anti-Flag and anti-C-MYC antibodies. Mw, molecular weight. (C-J) Bright-field images of the indicated control embryos and embryos injected with a subdose of MOs targeting wnt11f2/5b (5 and 1 ng). Lateral view. Lines with double arrows indicate posterior body length. Lines of the same color are equal in length. (K) Average posterior body length in embryos shown in C-J, from two independent experiments (represented by different color dots), with the number of embryos indicated. P values in different colors correspond to the embryos with posterior body length marked by the line of that color. Data are mean +- SEM. #, P > 0.05; **, P < 0.01; ****, P < 0.0001; unpaired Student's t test.

Supportive validation

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Experimental details: Figure 4. Acr3 is a component of the Fps1 complex that regulates Fps1 in response to arsenicals. (A) As(V) treatment induces Fps1 closure in an acr3 Delta mutant. Rgc2-HA and Fps1-Myc were coexpressed (from p3151 and p3121, respectively) in a WT strain (DL3187), or an acr3 Delta mutant (DL4287). The strains were treated with 1 mM As(III) or 3 mM As(V) for the indicated times and processed for co-IP of Rgc2 with Fps1 using anti-Myc antibodies, as in Fig. 1 . (B) Acr3 associates with Fps1. A WT strain (DL3187) coexpressing Fps1-Myc (p3121) and Acr3-HA (p3470) or expressing only one protein was subjected to co-IP analysis using anti-HA antibodies. (C and D) BiFC analysis of Acr3 with known components of the Fps1 complex. An rgc1 Delta rgc2 Delta hog1 Delta strain (C; DL3219) coexpressing CFPC-Fps1 (p3216) and Acr3-CFPN (p3585) was visualized under a UV light source to reveal fluorescence complementation or under visible light (DIC). An fps1 Delta strain (D; DL3226) coexpressing CFPC-Rgc2 (p3584) and Acr3-CFPN (p3585) was visualized as above. Representative micrographs are shown. (E) Pretreatment of WT cells with As(III), but not As(V), blocked As-biotin binding to Acr3-HA. WT strain (DL3187), expressing Acr3-HA (p3470) was pretreated with 1 mM As(III) or 3 mM As(V) for 20 min prior to treatment with 10 muM As-biotin for 10 min. Extracts were subjected to affinity pull-down with SA beads prior to SDS-PAGE and immunoblot analysis for Acr3-HA. (F) Acr3 is a MAs(III) target. An mtq2

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Experimental details: Figure 7. Cysteine residues in the Acr3 cytoplasmic loop #4 are modified by MAs(III) and are important for the regulation of Fps1. (A) Effect of Acr3 cytoplasmic loop #4 deletion on Fps1 closure in response to As(V) treatment. An acr3 Delta strain (DL4287) cotransformed with plasmids expressing Fps1-Myc (p3121), Rgc2-HA (p3151) and either ACR3 (p3587) or ACR3-Delta307-334 (p3588) were treated with 3 mM As(V) for the indicated times and processed for co-IP of Rgc2 with Fps1 using anti-Myc antibodies. (B) The three cysteine residues within Acr3 loop #4 account for its As-biotin binding. An acr3 Delta strain (DL4287) expressing the indicated form of Acr3 was treated with 10 uM As-biotin for 10 min. Extracts were subjected to affinity pull-down with SA beads prior to SDS-PAGE and immunoblot analysis for Acr3-HA. Plasmids expressed one of the following ACR3 alleles: ACR3-HA (p3470), acr3-C169S-HA (p3591), acr3-C333S-HA (p3592), acr3-C316S , C318S-HA (2C/S; p3593), or acr3-C316S , C318S , C333S-HA (3C/S; p3594). (C) Cys-to-Ser mutations within Acr3 loop #4 allow closure of Fps1 in response to As(V) treatment. An acr3 Delta strain (DL4287) was cotransformed with plasmids expressing Fps1-Myc (p3121), Rgc2-HA (p3151) and one of the following: ACR3 (p3587), acr3-C316S , C318S (p3589), or acr3-C316S , C318S , C333S (p3590). Strains were either treated with 3 mM As(V) for 10 min or untreated (C) and processed for co-IP of Rgc2 with Fps1. (D) As(V) challenge. As(V) is reduced to As(III),

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Experimental details: Figure 2 Disease-causing mechanisms of BEST1 mutations. ( a ) Bar chart showing population steady-state current densities at 100 mV for transiently expressed BEST1 WT and mutants in HEK293 cells at 1.2 muM [Ca 2+ ] i ; n = 5-6 for each point. * P = 4 x 10 -5 (A10T), 7 x 10 -4 (R218H), 2 x 10 -3 (L234P), 3 x 10 -2 (A243T), 2 x 10 -4 (Q293K), 2 x 10 -3 (D302A), compared to WT, respectively, and # P = 6 x 10 -4 for A243T compared to untransfected cells, using two-tailed unpaired Student t test. ( b ) WT or mutant BEST1-YFP-His was co-transfected with WT BEST1-CFP-Myc to HEK293 cells, and detected by immunoblotting directly in cell lysate (input) or after co-immunoprecipitation. The full-length blots are shown in Fig. S6 . All error bars in this figure represent s.e.m. ( c ) Ribbon diagram of two oppositely facing (144deg) protomers of a BEST1 pentamer are shown with the extracellular side on the top. The side chains of critical residues are in red. ( d ) Location of the patient mutations in relationship to the channel pore, as viewed from the side. A10 (red), Q293 (green) and D302 (blue) are colored differently. R218, L234 and A243 are colored by atoms. ( e ) Possible interactions of the mutated residues. Each monomeric unit is drawn by a different color and the mutated residues are colored in magenta. Coordination bonds and possible hydrogen bonds are illustrated by dotted black and yellow lines, respectively. ( f ) The effect of replacing R218 with H. Possible conformations of

Supportive validation

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Experimental details: Fig. 6 Expression of recombinant MsLAC1 protein and oxidation of sinapyl alcohol by purified recombinant protein. a The protein was expressed in P. pastoris and analyzed by Native PAGE and Western blot. After methanol induction samples of culture medium were collected at daily intervals, cleared by centrifugation, and 10 mul of supernatant were filtered and loaded. The zymogram was stained with 10 mM ABTS. Western blot was developed with anti-MYC antibody. M: PageRular Prestained Protein Ladder, 10-170 kDa. C: Crude extract from fermentation broth of P. pastoris transformed with pPICZalphaAfeh, 8 days after induction. Sinapyl alcohol (final concentration 1 mM) was incubated with 0.002 U recombinant MsLAC1 protein in 50 mM acetate buffer, pH 3.0. b Spectra of reaction mixtures (250 nm - 650 nm) were recorded at indicated time intervals (the insert indicates the control with substrate only). c Time course of absorption change at 280 nm. The initial absorbance at 280 nm was set as 100% individually for the calculation of absorbance decrease with purified protein and water control

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Experimental details: Figure 3 Menin and MYC colocalize on chromatin. ( a ) Venn diagram of the ChIP-seq data showing the overlap of genes bound by MYC and Menin. ( b ) Motif enrichment analysis of the sequences at gene promoters from MYC and Menin ChIP-seq data. Consensus E-box sequences (CACGTG) were significantly enriched from both MYC ( P -value

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Experimental details: Fig. 4 VHL modulates SARS-CoV nsp16 ubiquitination. (A) Left panels, HEK-293T cells were transfected with Myc-ubiquitin, Flag-nsp16 and an increasing amount of HA-VHL (1.0 and 2.0 mug). After 36 h transfection, the cells were collected and denatured in 1% SDS lysis buffer, and the cell lysate were diluted with lysis buffer until the concentration of SDS decreased to 0.1%. The cell extracts were used for co-immunoprecipitation with an anti-Flag antibody. The immunoprecipitates were detected by an anti-Myc or anti-Flag antibody. Right panels, assays were performed as in the left panels. SARS-CoV nsp14 as a negative control and Dvl2 as a positive control. (B) The HA-tagged ubiquitin mutants, Flag-nsp16 and Myc-VHL were transfected into HEK-293T cells. After 36 h, re-IP assays were performed with anti-Flag antibody. The immunoprecipitates were analyzed with an anti-HA or anti-Flag antibody. (C) The HA-ubiquitin, Flag-nsp16 or Flag-nsp14 or Flag-Dvl2, control (PLKO.1) or VHL RNAi plasmids (1# or 2#) were transfected into HEK-293T cells. After 24 h, the cells were treated with puromycin (1 mug/ml) for another 24 h and then lysed for re-IP assays with anti-Flag antibody. The immunoprecipitates were analysed with the anti-HA or anti-Flag antibody (upper panels).

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Experimental details: Figure 3 CCN1 enhances the function of hepatic stellate cells in promoting the viability of HCCs. (A) Western blot analysis was used to detect expression levels of CCN1 in the original CM and 10X concentration CM collected from LX-2 cells infected with AdCCN1 or AdRFP. Expression levels were also detected following treatment with 2.5 and 5 mu g. HepG2 cells were cultured with or without CM. (B) Viability of HepG2 cells, analyzed using MTT assays. * P

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Experimental details: Figure 4 Residues 40-49 Form an NTE-Specific Kinase Autoinhibitory Module (A) In vitro kinase assay of MYC-GFP-Mps1 constructs. Coomassie blue staining indicated equal loading. Quantification of Mps1 autophosphorylation is shown; data are means +- SEM; n = 3. * p < 0.05 and ** p < 0.01. (B) Immunoblotting with the indicated antibodies of the various MYC-Mps1 mutant proteins. (C) MYC immunoprecipitates from cells expressing the indicated MYC-Mps1 constructs were resolved by SDS-PAGE and immunoblotted as shown. (D) Cells were treated as in (C), enriched in mitosis with nocodazole, and immunoblotted as indicated. See also Figures S4 and S5 .

Supportive validation

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Experimental details: Figure 8 (A) Solid agar plates depicting the growth of yeast cellsthatexpress human Bax protein and cells that do not contain Bax, in minimalmedium containing either glucose (SD) or galactose (SG). (B) Growthof one set of clones from the strains, as in (A), in liquid minimalmedium containing galactose (SG); the OD 600 values at eachtime point represent the average of three independent experiments(* P < 0.01; one-tailed Student's t -test). (C) Western blot of total cellular proteins obtainedafter lysis of cells that do not express Bax (BC300::--) anddo express c -myc-tagged Bax (BC300::Bax), probedwith a c -myc antibody (Thermo Scientific, MA 1-980),and a beta-actin antibody (Proteintech, 60008-1-Ig) as a loadingcontrol, beta-actin being a housekeeping gene. (D) Staining ofcells that express BC300::-- and BC300::Bax, with Phloxine B,under the fluorescent microscope. The left-hand panels show cellsin phase contrast microscopy. (E) Rescue from the block in growthof yeast cells that express human Bax, by methoxy-stilbenes, at 10muM. The data represent mean +- SD of four independent experimentswhere optical density at 600 nm (OD 600 ) was measured foreach cell culture.

Supportive validation

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Experimental details: Figure 1 VDR interacts with NLRP3. (A) Mass spectrometry analysis of NLRP3 and VDR peptides after immunoprecipitation with Flag to pull down NLRP3-associated proteins in Flag-NLRP3-overexpressing HEK293T cells. (B) HA-VDR was co-expressed with Flag-tagged NLRP3 in HEK293T cells; proteins were immunoprecipitated and analyzed by immunoblotting. Whole-cell lysates are shown as the input. (C) LPS-primed BMDMs were unstimulated or stimulated with nigericin for 30 min. Cell lysates were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies. (D) Immunofluorescent staining for VDR and NLRP3 in LPS-primed BMDMs treated with or without nigericin. Scale bar, 10 mum. (E) Purified GST-VDR was incubated with purified His-NLRP3 for 2 h. His-NLRP3-Flag bound to GST-VDR was pulled down by glutathione beads and subjected to immunoblot analysis. (F) Wild-type or mutant NLRP3 (PYD, NACHT, or LRR) and HA-VDR were expressed in HEK293T cells, immunoprecipitated, and analyzed by immunoblotting. (G) Wild-type or mutant VDR (DBD or LBD) and Flag-NLRP3 were expressed in HEK293T cells, immunoprecipitated, and analyzed by immunoblotting.

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Experimental details: Figure 5 Vitamin D receptor inhibits NLRP3 deubiquitination. (A) HEK293T cells were transfected with the indicated vectors. NLRP3 ubiquitination was analyzed. (B) Immunoblot analysis of NLRP3 after treatment of LPS-primed BMDMs (wild-type and Vdr -/- ) with CHX (1 muM) for the indicated time. (C) HEK293T cells expressing HA-BRCC3 and Myc-NLRP3 PYD, NACHT, or LRR were co-transfected with VDR as indicated. Myc-NLRP3 immunoprecipitants were analyzed for ubiquitination. (D) HEK293T cells expressing Flag-NLRP3, HA-BRCC3, and HA-Ubiquitin K63 or K48 were collected, and the cell lysates were immunoprecipitated with the anti-Flag antibody to detect ubiquitination.

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Experimental details: Fig. 4 Combinatorial depletion of different sets of multiple proteins. a Schematic outline of the multi-protein targeting DiPD system. Multiple POIs (proteins of interest, POI1-3) are targeted by distinct protein specific targeting modules (Nanobodies, Nbs), which are embedded in the DiPD system. Upon induction with rapamycin the POIs are ubiquitinated and degraded. E3 ubiquitin E3 ligase. b Triple depletion of endogenous LMNA/C, PCNA and GFP-CENPA proteins. GFP-CENPA cells were transfected with DiPD constructs targeting LMNA/C, PCNA and GFP. Proteins were detected by immunostaining or GFP fluorescence after rapamycin treatment. A triple depletion of targeted proteins could be observed in the presence of rapamycin. c Principle of combinatorial light and drug-induced depletion of two independent proteins (POI1 and POI2). d Combinatorial depletion of a nucleoplasmic (GFP-CXXC4, in green) and a nuclear envelope protein (LMNA/C, in magenta). Cells with the light and drug inducible protein depletion tools express DsRed as marker (in gray). Scale bars are 10 um. Extended versions of this figure including uninduced controls are shown in Supplementary FigS. 10 and 11 .

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Experimental details: Figure 6 BET inhibition as a single agent or in combination with CHK1i/SRA737 does not decrease the MYC protein. Saos2 cells were treated with controls (media and vehicle), as well as single-agent and combination therapy involving BETi/OTX-015 (0.31 uM) and/or CHK1i/SRA737 (3.75 uM) for 4 or 16 h. Concentrations were obtained from data in Table 2 a. Vinculin serves as the loading control. This was performed once. OTX = BETi/OTX-015 and SRA = CHK1i/SRA737.